It is widely known that the hippocampus is critically involved in acquisition and retrieval of episodic memory and the dentate gyrus is the central information processor for sensory inputs to the hippocampus. The mossy cells in the dentate hilus are the only glutamatergic non-principal cells in the hippocampus and are mainly located in the ventral hilus. Mossy cells receive glutamatergic inputs from the granule cells of the dentate gyrus and the pyramidal cells of area CA3, and provide massive recurrent connections to dentate granule cells along the longitudinal axis of the hippocampus. Therefore, mossy cells have been suggested to play a significant role in dentate functions such as memory formation. However, the in vivo contribution of mossy cells to the dentate network has not been explored yet. [unreadable] To address the role of this mossy cell-mediated dentate gyrus network in vivo, we have generated hippocampal mossy cell-restricted NMDA receptor subtype 1 knockout mice (mossy cell NR1-KO mice or mutant), by crossing a mossy cell-restricted Cre line (Cre #4688) and floxed NR1 line. Using these mice, Seiichiro Jinde and others have been working on the behavioral research to elucidate in vivo NR function of mossy cells in the acquisition and retrieval of context specificity in an associative memory, such as the ability to distinguish two similar input patterns (pattern separation). [unreadable] To study pattern separation, these mutants and their control littermates were subjected to one-trial context discrimination tasks using a contextual step-through avoidance task and contextual fear conditioning. Three hours after one-trial avoidance training (0.12 mA foot shock), mutants showed a clear deficit in discriminating the conditioned context and the non-shocked context while the controls were able to distinguish the two contexts. On the other hand, the mutants showed no impairment in discriminating conditioned and no-shock contexts 3 hours after one-trial contextual fear conditioning when strong foot-shock intensity was used. Interestingly, our previous study showed that CA3 NR1-KO mice were impaired in both tasks (Cravens et al., 2006). Thus, while mossy cell NR1 could contribute to contextual pattern separation in the dentate, this phenomenon may be functionally distinct from that observed in CA3. Furthermore, our preliminary results suggested that expression of immediate early genes in the dentate granule cells following the kainate injection is increased in the mutants, suggesting dentate granule cell hyperexcitability caused by ablation of mossy cell NR1. Taken together, these results identify a crucial role for mossy cell NR1 in context acquisition, which could be through the regulation of dentate granule cell excitability. Veronika Zsiros is now setting up slice physiology experiments to evaluate the excitability of the dentate granule cells in physiological conditions. [unreadable] We have also generated another mutant line with transgenic expression of floxed-diphtheria toxin receptor (DTR) under control of the CaMKII-alpha promoter, which limits expression to neurons of the forebrain. After crossing with the Cre-expressing mouse strain, Cre#4688, the double-transgenic mutants express DTR on the surface of Cre-expressing dentate mossy cells. Since expression of DTR allows binding of the B subunit of DT and subsequent receptor-mediated endocytosis, i.p. injection of diphtheria toxin (DT) induces the ablation of the Cre-expressing cell. Our preliminary study showed that at least 70% of mossy cells were ablated after DT injection. Histological, electrophysiological and behavioral analysis is in progress to evaluate the consequence of hilar mossy cell ablation in vivo.